The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.     

Development of indole/indazole-aminopyrimidines as inhibitors of c-Jun N-terminal kinase (JNK): Optimization for JNK potency and physicochemical properties

A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC₅₀ of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties.     

Fish oil supplementation for two generations increases insulin sensitivity in rats

We investigated the effect of fish oil supplementation for two consecutive generations on insulin sensitivity in rats. After the nursing period (21 days), female rats from the same prole were divided into two groups: (a) control group and (b) fish oil group. Female rats were supplemented with water (control) or fish oil at 1 g/kg body weight as a single bolus for 3 months. After this period, female rats were mated with male Wistar rats fed on a balanced chow diet (not supplemented). Female rats continued to receive supplementation throughout gestation and lactation periods. The same treatment was performed for the next two generations (G1 and G2). At 75 days of age, male offspring from G1 and G2 generations from both groups were used in the experiments. G1 rats did not present any difference with control rats. However, G2 rats presented reduction in glycemia and lipidemia and improvement in in vivo insulin sensitivity (model assessment of insulin resistance, insulin tolerance test) as well as in vitro insulin sensitivity in soleus muscle (glucose uptake and metabolism). This effect was associated with increased insulin-stimulated p38 MAP kinase phosphorylation and lower n-6/n-3 fatty acid ratio, but not with activation of proteins from insulin signaling (IR, IRS-1 and Akt). Global DNA methylation was decreased in liver but not in soleus muscle. These results suggest that long-term fish oil supplementation improves insulin sensitivity in association with increased insulin-stimulated p38 activation and decreased n-6:n-3 ratio in skeletal muscle and decreased global DNA methylation in liver.     

Implicit Motivational Impact of Pictorial Health Warning on Cigarette Packs

Objective

The use of pictorial warning labels on cigarette packages is one of the provisions included in the first ever global health treaty by the World Health Organization against the tobacco epidemic. There is substantial evidence demonstrating the effectiveness of graphic health warning labels on intention to quit, thoughts about health risks and engaging in cessation behaviors. However, studies that address the implicit emotional drives evoked by such warnings are still underexplored. Here, we provide experimental data for the use of pictorial health warnings as a reliable strategy for tobacco control.

Methods

Experiment 1 pre-tested nineteen prototypes of pictorial warnings to screen for their emotional impact. Participants (n = 338) were young adults balanced in gender, smoking status and education. Experiment 2 (n = 63) tested pictorial warnings (ten) that were stamped on packs. We employed an innovative set-up to investigate the impact of the warnings on the ordinary attitude of packs’ manipulation, and quantified judgments of warnings’ emotional strength and efficacy against smoking.

Findings

Experiment 1 revealed that women judged the warning prototypes as more aversive than men, and smokers judged them more aversive than non-smokers. Participants with lower education judged the prototypes more aversive than participants with higher education. Experiment 2 showed that stamped warnings antagonized the appeal of the brands by imposing a cost to manipulate the cigarette packs, especially for smokers. Additionally, participants’ judgments revealed that the more aversive a warning, the more it is perceived as effective against smoking.

Conclusions

Health warning labels are one of the key components of the integrated approach to control the global tobacco epidemic. The evidence presented in this study adds to the understanding of how implicit responses to pictorial warnings may contribute to behavioral change.     

Coping with potential bi‐parental inbreeding: limited pollen and seed dispersal and large genets in the dioecious marine angiosperm Thalassia testudinum

The high prevalence of dioecy in marine angiosperms or seagrasses (>50% of all species) is thought to enforce cross‐fertilization. However, seagrasses are clonal plants, and they may still be subject to sibling‐mating or bi‐parental inbreeding if the genetic neighborhood is smaller than the size of the genets. We tested this by determining the genetic neighborhoods of the dioecious seagrass Thalassia testudinum at two sites (Back‐Reef and Mid‐Lagoon) in Puerto Morelos Reef Lagoon, Mexico, by measuring dispersal of pollen and seeds in situ, and by fine‐scale spatial autocorrelation analysis with eight polymorphic microsatellite DNA markers. Prevalence of inbreeding was verified by estimating pairwise kinship coefficients; and by analysing the genotypes of seedlings grown from seeds in mesocosms. Average dispersal of pollen was 0.3–1.6 m (max. 4.8 m) and of seeds was 0.3–0.4 m (max. 1.8 m), resulting in a neighborhood area of 7.4 m² (range 3.4–11.4 m²) at Back‐Reef and 1.9 (range 1.87–1.92 m²) at Mid‐Lagoon. Neighborhood area (Na) derived from spatial autocorrelation was 0.1–20.5 m² at Back‐Reef and 0.1–16.9 m² at Mid‐Lagoon. Maximal extensions of the genets, in 19 × 30 m plots, were 19.2 m (median 7.5 m) and 10.8 m (median 4.8 m) at Back‐Reef and Mid‐Lagoon. There was no indication of deficit or excess of heterozygotes nor were coefficients of inbreeding (F_IS) significant. The seedlings did not show statistically significant deficit of heterozygotes (except for 1 locus at Back‐Reef). Contrary to our expectations, we did not find evidence of bi‐parental inbreeding in this dioecious seagrass with large genets but small genetic neighborhoods. Proposed mechanisms to avoid bi‐parental inbreeding are possible selection against homozygotes during fecundation or ovule development. Additionally, the genets grew highly dispersed (aggregation index Ac was 0.09 and 0.10 for Back‐Reef and Mid‐Lagoon, respectively); such highly dispersed guerrilla‐like clonal growth form likely increases the probability of crossing between different potentially unrelated genets.     

Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein

Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein. CHIKV structural proteins are expressed from a subgenomic RNA synthesized in infected cells. Here we have developed CHIKV trans-replication systems, where replicase expression and RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein and template RNAs, which are recognized by CHIKV replicase and encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates and synthesized large amounts of subgenomic RNA in several cell lines. This system was used to create tagged versions of ns proteins including nsP1 fused with enhanced green fluorescent protein and nsP4 with an immunological tag. Analysis of these constructs and a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional and maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA and wild type or tagged versions of CHIKV replicases, formation of characteristic replicase complexes (spherules) was observed. Analysis of mutations associated with noncytotoxic phenotype in CHIKV replicons showed that a low level of RNA replication is not a pre-requisite for reduced cytotoxicity. The CHIKV trans-replicase does not suffer from genetic instability and represents an efficient, sensitive and reliable tool for studies of different aspects of CHIKV RNA replication process.     

Differences in the Access to Sterilization between Women Living and Not Living with HIV: Results from the GENIH Study,Brazil

BackgroundIn many countries, young women of reproductive age have been especially affected by the HIV epidemic, which have fostered research to better understand how HIV infection influences and shapes women´s fertility and reproductive and sexual decisions. In Brazil, few studies have focused on the impact of the HIV epidemic on contraceptive choices among women living with HIV (WLHIV).ObjectiveThis study evaluates the impact HIV infection may have in the access to female sterilization in Brazil, using a time-to-event analysis.MethodsA cross-sectional quantitative study (GENIH study) was conducted between February 2013 and April 2014 in the city of São Paulo, comparing two probabilistic samples of 975 WLHIV and 1,003 women not living with HIV (WNLHIV) aged 18 to 49. Sexual and reproductive data was collected retrospectively in order to reconstruct women''s reproductive trajectories. Given the objectives of this study, the analysis was restricted to women with parity one or more and, in case of WLHIV, to those sterilized after HIV diagnosis and not infected through vertical transmission. The final sample analysis included 683 WNLHIV and 690 WLHIV. A series of multivariable-adjusted Cox models estimated the probability of being sterilized after HIV diagnosis, compared with WNLHIV. Models were adjusted for schooling, race/color, and stratified by parity at last delivery (1–2, 3+). Hazard ratios were calculated for female sterilization, and separately for interval and postpartum procedures (performed in conjunction with caesarean section or immediately after vaginal delivery). Additionally, information regarding unmet demand for female sterilization was also explored.FindingsNo statistical difference in the overall risk of sterilization between WLHIV and WNLHIV in the two parity-related groups is observed: HR = 0.88 (0.54–1.43) and 0.94 (0.69–1.29), respectively, among women with 1–2 children and those with three and more. However, significant differences regarding the impact of HIV infection at sterilization are observed depending on the timing and the type of sterilization procedure. The probability of obtaining an interval sterilization is significantly lower for WLHIV compared to those not living with HIV. The reverse occurs regarding postpartum sterilization. Although sterilization is mainly performed in conjunction with caesarean section in Brazil, it is evident that caesarean sections are not the sole factor increasing the risk of sterilization among WLHIV.ConclusionThe results indicate barriers in the access to services offering interval sterilization for WLHIV and certain facilitation in obtaining the procedure in conjunction with caesarean section. Health policy makers at local and national levels should promote institutional changes in order to facilitate access to interval sterilization and to confront the sensitive discussion of WLHIV’s eligibility for postpartum sterilization. It is also urgent to increase access to a wider range of contraceptive methods for WLHIV and promote dual method protection strategies. Moreover, since condom use may decrease in the future in the context of the preventive effect of antiretroviral therapy, promoting dual methods will expand the choices regarding the reproductive rights of women living with HIV.     

Conditional restoration and inactivation of Rbm47 reveal its tissue‐context requirement for viability and growth

Rbm47 encodes a RNA binding protein that is necessary for Cytidine to Uridine RNA editing. Rbm47^gt/gt mutant mice that harbor inactivated Rbm47 display poor viability. Here it was determined that the loss of Rbm47^gt/gt offspring is due to embryonic lethality at mid‐gestation. It was further showed that growth of the surviving Rbm47^gt/gt mutants is impaired. Rbm47 is expressed in both the visceral endoderm and the definitive endoderm. Using the utility of the switchable FlEx gene‐trap cassette and the activity of Cre and FLP recombinases to generate mice that conditionally inactivate and restore Rbm47 function in tissue‐specific manner, it was demonstrated that Rbm47 function is required in the embryo proper, and not the visceral endoderm, for viability and growth. genesis 54:115–122, 2016. © 2016 Wiley Periodicals, Inc.     

Roles of the ClpX IGF loops in ClpP association,dissociation, and protein degradation

IGF‐motif loops project from the hexameric ring of ClpX and are required for docking with the self‐compartmentalized ClpP peptidase, which consists of heptameric rings stacked back‐to‐back. Here, we show that ATP or ATPγS support assembly by changing the conformation of the ClpX ring, bringing the IGF loops closer to each other and allowing efficient multivalent contacts with docking clefts on ClpP. In single‐chain ClpX pseudohexamers, deletion of one or two IGF loops modestly slows association with ClpP but strongly accelerates dissociation of ClpXP complexes. We probe how changes in the sequence and length of the IGF loops affect ClpX–ClpP interactions and show that deletion of one or two IGF loops slows ATP‐dependent proteolysis by ClpXP. We also find that ClpXP degradation is less processive when two IGF loops are deleted.     

Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection

Larval and adult mosquitoes mount immune responses against pathogens that invade their hemocoel. Although it has been suggested that a correlation exists between immune processes across insect life stages, the influence that an infection in the hemocoel of a larva has on the immune system of the eclosed adult remains unknown. Here, we used Anopheles gambiae to test whether a larval infection influences the adult response to a subsequent bacterial or malaria parasite infection. We found that for both female and male mosquitoes, a larval infection enhances the efficiency of bacterial clearance following a secondary infection in the hemocoel of adults. The adults that emerge from infected larvae have more hemocytes than adults that emerge from naive or injured larvae, and individual hemocytes have greater phagocytic activity. Furthermore, mRNA abundance of immune genes—such as cecropin A, Lysozyme C1, Stat‐A, and Tep1—is higher in adults that emerge from infected larvae. A larval infection, however, does not have a meaningful effect on the probability that female adults will survive a systemic bacterial infection, and increases the susceptibility of females to Plasmodium yoelii, as measured by oocyst prevalence and intensity in the midgut. Finally, immune proficiency varies by sex; females exhibit increased bacterial killing, have twice as many hemocytes, and more highly express immune genes. Together, these results show that a larval hemocoelic infection induces transstadial immune activation—possibly via transstadial immune priming—but that it confers both costs and benefits to the emerged adults.